With the recent generation of comprehensive genome-wide maps of diverse epigenetic modifications in multiple cell types and disease states there is now a pressing need to move beyond descriptive epigenomics. Crucially, development of functional epigenomics tools to deliberately and precisely change specific epigenetic modifications is required, in order to interrogate the role of epigenetic marks in genome regulation in myriad cell types and model systems. Herein we propose the generation of novel state-of-the-art programmable DNA- binding proteins that ferry epigenome-modifying activity precisely to desired target loci in the genome. By linking a sequence specific DNA-binding domain (DBD) engineered from a Transcription Activator-Like Effector (TALE) protein backbone to an epigenome modifier (epiTALE) we will achieve highly precise epigenome engineering. We have chosen to program DNA methylation (DNAme) changes at a wide range of loci in the human genome encompassing different functional chromatin states, both open chromatin and heterochromatin. We will rapidly construct highly specific TALE-DBDs able to recognize unique ~20 bp sequences in the genome, and linked to either the catalytic domain of DNA methyltransferase 3a (DNMT3A) for de novo DNAme, or VP64 or the TET1 for targeted DNA demethylation. Our specific hypothesis is that linking custom TALE- DBDs to chromatin modifiers will enable site-specific modulation of the DNAme state in a broad range of chromatin states in the human genome, such as enhancers and promoters. First, we propose to design a focused panel of epiTALEs to target different chromatin states in IMR90 fibroblast cells, including active/inactive enhancers and promoters. Combined genomic analyses will be utilized to comprehensively assess the efficacy and activity of epiTALEs for epigenome engineering, including identification of epiTALE binding sites by ChIP-Seq, MethylC-Seq whole-genome bisulfite sequencing, and RNA-Seq. Second, we will spatio-temporally program the expression of epiTALEs using inducible systems, in which we can dynamically control (pulse and chase) the incorporation and the erasure of DNAme in the cells, following the spatial and temporal changes in targeted DNAme by high-throughput targeted bisulfite sequencing. Third, we present clinically relevant cell-type specific applications of these epigenome engineering tools; inducing locus specific modulation of DNAme to correct the aberrant epigenetic signatures found in breast cancer and induced pluripotent stem cells. This study will provide unprecedented insights into elusive questions in epigenomics, such as spreading of DNAme from a given nucleation point and the spatio-temporal dynamics of epigenetic memory. Our research will provide innovative molecular tools to assess the functional outcome of epigenetic perturbation in any sequence of interest in the genome and to correct aberrant epigenetic signatures identified in diseased or reprogrammed cells. Overall, this project aims to develop novel molecular tools for epigenome engineering that will constitute a major advance in the nascent field of functional epigenomics.